Studied on low molecular weight chitinase RS-9K

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Chitinase is the chitin hydrolytic enzyme. Since chitin is one of the major components of the cell wall of fungi, chitinase is thought to play the important role in the defense system of plants. To reveal the physiological role of chitinase, I studied chitinase of rice plant.Novel chitinase was purified from rice seeds by ion-exchange chromatography, gel filtration and reversed phase chromatography. The molecular weight of the chitinase isolated was 8,902 determined by TOF mass spectrometry, revealing one of the smallest enzymes. I was designated as chitinase RS-9K.Chitinase RS-9K was included as a small amount component in rice seed. Chitinase 9K DNA was cloned from rice genomic DNA and expressed by several expression systems. Baculovirus expression system, pET expression system in E.coli And pMAL expression system in E.coli weren't successful. By using pGEX expression system, recombinant chitinase RS-9K was obtained whereas it was not active. It was also difficult to reactivate recombinant chitinase RA-9K in some trials by means of reduction and reoxidation. Finally, the expression by yeast (pichia) was performed and the Active form of the recombinant enzyme was obtained.